Transient receptor potential melastatin 8 (TRPM8) is a cation channel expressed in a small subpopulation of sensory neurons, which detect innocuous cooling and mostly lack characteristics of nociceptors. Whether TRPM8 normally contributes to noxious cold sensing is debatable, as few TRPM8-positive neurons coexpress nociceptive markers. TRPM8 is a promising analgesic target. TRPM8 agonists cause analgesia in chronic pain states, by TRPM8-expressing afferents gating-out a wide range of hypersensitive pain responses in the spinal cord. TRPM8 antagonists may attenuate exaggerated responses to mild cooling during chronic pain but will not achieve generalized analgesia, as they can only block cool detection, not the clinically problematic hypersensitivity to other sensory modalities. Biochemical and physiological properties of TRPM8, the chemistry of TRPM8 ligands, intracellular modulation of TRPM8, and the neurobiological consequences of TRPM8 activation are discussed, with a view to future improvements in therapeutic targeting.

Only just over 10 years ago, the TRPM8 channel was cloned by several independent groups as the mediator of cool and menthol responsiveness in sensory ganglia and as an unknown transcript from prostate that was up-regulated in cancer [1–3]. Since that time, evidence has steadily and consistently accumulated to confirm a key physiological role of TRPM8 in the detection of innocuous cool. In addition, there is increasing interest in the channel as a potential target for new analgesics, especially in the context of chronic pain states that respond poorly to currently available therapeutics.

The TRPM8 channel is a nonselective cation channel assembled as a homotetramer of the 1104 amino acid protein, which individually has a molecular weight of around 128 kDa. The monomer has extended intracellular N- and C-termini and six transmembrane domains with a loop between domains 5 and 6 that is thought to contribute to the channel pore. Various reports implicate the C- or N-terminal segments in subunit assembly [4–6]. The C-terminal segment contains a short TRP box motif that is highly conserved throughout the family, including residues 1005 and 1009, involved in binding the allosteric potentiator PIP2 (phosphatidylinositol 4,5-bisphosphate), and is also necessary for agonist responsiveness and setting the cool temperature response threshold [2,3,7–9]. The initial part of the N-terminal segment has also been implicated in moderating responsiveness to cool and menthol [10].

Chemical agonists that activate TRPM8 include menthol and the synthetic compound icilin, both of which evoke a sensation of coolness. However, differences in channel residues involved in their recognition indicate that the two agonists bind to distinct sites, consistent with evidence that activation by icilin or cooling but not by menthol shows clear pH dependence and that icilin but not menthol action appears to require an increase in intracellular Ca2 + levels [11,12]. Mutation of residues 745, 842 and 856 in transmembrane domain 2 and the loop between domains 4 and 5 disrupts menthol activation whereas residues 799, 802 and 805 in transmembrane domain 3 are required for icilin effects [12–15]. TRPM8 also shows voltage sensitivity, with residues 842 and 856 probably contributing to voltage sensing [14,16,17]. Interactions between the different modes of activation are observed in that agonists not only increase open probability and conductance of the channel but also shift both its thermal threshold and voltage sensitivity toward more normal physiological levels [14,16–19]. The overall responses of a cell expressing TRPM8 to its activating stimuli will be further influenced by the effects of other ion channels on membrane potential and by signals from other receptors/intracellular signaling cascades.

TRPM8 has a highly selective tissue distribution. Apart from primary somatosensory neurons and prostate tissue (from where it was cloned), it is expressed at only low levels in other tissues. These include visceral afferents, bladder, vascular smooth muscle, stomach, liver, colon, lung and airways, sperm, and some primary tumors [20–28]. In the nervous system, TRPM8 is strongly expressed in trigeminal (TG) and dorsal root ganglia (DRG) and has subsequently been observed in autonomic ganglia [29–31]. However, there appears to be extremely limited TRPM8 expression within the central nervous system (CNS) other than that associated with afferent terminals [32]. This idea was fully corroborated by observations on mutant mice expressing green fluorescent protein (GFP) under the control of the TRPM8 promoter [33,34]. Furthermore, only 5-10% of DRG and TG neurons display the responsiveness to mild cooling (threshold around 25 °C), menthol and icilin that is characteristic of TRPM8, emphasizing the highly selective outcome to be expected from any intervention that might usefully target the channel. Early observations indicated that these neurons were small in diameter and distinct from those expressing classical nociceptive markers such as calcitonin gene-related peptide (CGRP), TRPV1, or binding sites for the lectin IB4 [3]. Most TRPM8-positive sensory neurons appear to be C fibers staining for peripherin, with a minor group of Aδ fibers, whose small cell bodies stain for neurofilament antigens [2,33–36]. Extensive in situ hybridization experiments in DRG indicated a pattern of cellular expression almost entirely distinct from that of the nociceptive channels TRPV1 and TRPA1 [37]. Other studies combining immunohistochemical and alternative localization techniques indicate a modest degree of overlap [33, 234,38], and our own dual immunofluorescence data from TRPM8 and TRPV1 (Table 14.1) concur in finding TRPV1 expression in a very small proportion of TRPM8-positive cells. A recent study utilizing genetically targeted ablation of TRPV1- and TRPM8-expressing cells reinforced the idea of minimal overlap [39]. Technical issues such as differences in sensitivity and selectivity of the reagents and methods used may contribute to the range of findings reported. A further factor to consider is that the extent of overlap may differ between somatic and autonomic sensory neurons where phenotypic subdivisions may differ from those of somatic afferents [20,24,40]. Studies exploring functional responses of TRPM8 have also tried to explore the extent to which any overlap is significant.

When cloned TRPM8 is expressed in oocytes or fibroblasts, the cells are reported to show a temperature activation range from around 22-25 °C (threshold) to 8-10 °C (maximal) [2,3]. Correspondingly, native TG neurons that show Ca2 + elevation responses selectively to menthol have thermal activation thresholds around 25 °C [41]. DRG or TG neurons from TRPM8−/− mice show significantly reduced responses to cooling stimuli, which ranged in different studies from 22 °C through to 9 °C, and in C fiber firing induced by cooling from 32 °C to 2 °C in a skin-nerve preparation [42–45]. With such readouts of responses to imposed progressive cooling, it is not clear whether components of responses in the noxious temperature range are lacking in TRPM8−/− mice as well as those from innocuous temperatures. These data remain equivocal in deciding whether TRPM8 is involved in physiological cold pain under normal conditions.

A number of studies on cultured TG or DRG cells describe subpopulations responding to both mild cooling (generally through the range 25-17 °C) and to menthol that are considered to reflect TRPM8-expressing cells [46–49]. Some caution is needed, though, as menthol has only modest selectivity for TRPM8 over TRPA1 [50], which also displays sensitivity to reduced temperatures and is expressed in a subset of TRPV1-positive nociceptors [41,51]. A careful comparison of the temperature sensitivity of individual menthol-responsive/allyl isothiocyanate (TRPA1 agonist)-unresponsive TG neurons (likely to express TRPM8) and those activated by both menthol and allyl isothiocyanate (likely to express TRPA1) points to higher (innocuous range) temperature thresholds in the TRPM8 group but with substantial overlap [41].

Although menthol responsiveness in itself does not decisively implicate TRPM8, some studies have explored responsiveness of sensory neuron populations to menthol and capsaicin [48]. Neurons with dual responsiveness could be taken as evidence for functionally significant TRPM8 channels in nociceptors, but the debate remains equivocal because of menthol’s limited selectivity for TRPM8 over TRPA1. In addition, the electrophysiological responses of menthol-activated (putative TRPM8-positive) DRG neurons to mild cooling to 24 °C, involving tetrodotoxin-sensitive Na+ channels, appeared to be attenuated at the more intense cold level of 10 °C, whereas the intense cold-induced firing in neurons with tetrodotoxin-insensitive Na+ channels (putative nociceptors) remained robust [52].

The development of highly selective agonist and antagonist tools for TRPM8 should help to further elucidate the situation. An additional variable in experiments with cultured sensory neurons is the possibility of a change in phenotype relating to duration in vitro [34], a parameter that can differ considerably between studies and may also undergo some transition during chronic pain states [53].

C fiber recordings in rodents identify two distinct populations of cold-sensitive neurons [54]. The first of these comprises low temperature threshold, mechanoinsensitive, heat-insensitive afferents, sensitive to small reductions in skin temperature of as little as 2 °C, that are activated by menthol (10% topical) and by evaporative cooling due to acetone (i.e., nonnociceptive thermoreceptors that may express TRPM8). The second comprises high threshold cold-, mechano- and heat-sensitive nociceptive afferents, firing at 12 °C or below that are indifferent to menthol. These findings are consistent with a significant role for TRPM8 in innocuous cool detection but not cold pain in normal animals. Similar profiles are reported from microneurography experiments in normal human volunteers [55]. Very high concentrations of menthol (up to 40%) applied topically to the skin are perceived as noxious [56–58] [a situation mirrored by behavioral experiments in rodents [36]], but interpretative caution is needed as TRPM8 selectivity is uncertain at such concentrations. In the special circumstance of the cornea, highly sensitive cool thermoreceptors predominate, which respond vigorously to small reductions in temperature (as little as 0.5 °C reduction) and contain abundant TRPM8 [59].

Thermal place preference tests with TRPM8−/− mice indicate lack of sensation across the innocuous cooling temperature range [42–44,60]. Whereas colder temperatures (10 °C and below) showed return of temperature preference and cold plate paw withdrawal responses (10 °C and below) were normal in TRPM8−/− mice [42,44,45], some attenuation of thermal preference was seen, to a greater or lesser extent, at temperatures as low as 5 °C. Correspondingly, the flicking responses due to evaporative cooling from acetone, which can cool the skin to temperatures around 14-18 °C [43,61], were substantially reduced in TRPM8−/− mice [42,44] or by systemic administration of the high-affinity TRPM8 antagonist, PBMC [61]. As this temperature range corresponds to the loosely defined border between innocuous and noxious cooling in man [62] and the precise thermal consequences of differing laboratory protocols are uncertain, it is not clear that the test explicitly reflects cold pain as opposed to a response to innocuous cooling.

Intraplantar injection of the selective TRPM8 agonist icilin at high local concentrations (8 mM solution) can evoke flinching behavior and spinal cord c-Fos expression, which are reduced in TRPM8−/− mice [43–45,63]. Intraplantar injections of icilin or menthol at high concentrations, however, cause activation of a wide variety of sensory neurons of different types through apparently TRPM8-independent processes [64]. Furthermore, many agents when injected directly into the skin, presumably adjacent to sensory nerve terminals, can elicit nociceptive responses that they would not normally cause through other routes such as topical administration [65]. Although intraplantar icilin-evoked nocifensive behavior appears to involve TRPM8, it is not clear that this relates to physiological cold sensing.

As adaptive compensatory responses are possible in constitutive knockout animals, a targeted ablation strategy has also been investigated. In TRPM8 neuron-ablated mice (which express a diphtheria toxin (DTx) receptor transgene under the control of the TRPM8 promoter and were treated with DTx) results corroborated those in TRPM8−/− mice [39,60]. TRPM8 ablation abrogated behavioral responses to acetone-induced evaporative cooling and thermal preference through the innocuous cooling range of 30-10 °C [39,60]. However, avoidance of 0-10 °C cold surfaces and paw withdrawal/flinching to severe noxious cold were also attenuated in TRPM8 neuron-ablated mice, and this was to a greater extent than in TRPM8−/− mice [39,60]. Some of these data were obtained with a new sensitive forepaw-flinching assay, but the precise extent of temperature reduction reached in the forepaws may be affected by guarding behavior. Furthermore because the strategy ablates neuronal populations rather than individual candidate molecular mediators, additional proteins in subsets of TRPM8-expressing cells could be key to noxious cold sensing.

Preexposure to chemical agonists such as menthol markedly increases cold-induced Ca2 + entry in TRPM8-expressing oocytes or fibroblasts and menthol-sensitive TG cells, as well as cold-induced firing in a skin-nerve preparation [2,3,66], a phenomenon also reported with TRPA1 [67]. The effective temperature threshold for cellular responses to cooling can also be influenced by coexpressed K+ channels acting to hyperpolarize the membrane and oppose TRPM8-mediated depolarization. Both Kv1 and Kv7 family channels are coexpressed with TRPM8, notably in nociceptors where high K+ channel: modest TRPM8 expression ratios may drive the cold threshold into the noxious temperature range [66,68]. In contrast, low-threshold nonnociceptive cool-sensing afferents appear to have high ratios of TRPM8: K+ channel expression [59,66,68]. In addition, K2P TREK family channels, some of which are directly closed by temperature reductions, are present in subsets of TRPM8-positive cells [69] and may impact on their thermal sensitivity. Effects of cooling on A-type K+ channels and tetrodotoxin-sensitive or resistant Na+ channels may also modulate firing in cool thermoreceptors and cold nociceptors [52,70], whereas the notable resistance of Nav1.8 to cooling-induced desensitization is crucial for transmission in cold-sensitive afferents [71]. TRPM8 function (and indeed that of any other threshold-setting channels) is, of course, also subject to a variety of modulatory influences from intracellular signaling events (see later).

Chronic pain states of either inflammatory or neuropathic origin crucially involve central hypersensitivity that is brought about by neurochemical changes ensuing from maintained nociceptor firing [72]. This will manifest as exaggerated responses to noxious stimuli (hyperalgesia) and perception of normally innocuous stimuli as noxious (allodynia). This central resetting of excitability will most likely apply to thermal, mechanical, and cool sensory inputs, so in the case of TRPM8-mediated inputs a degree of cool allodynia would be entirely expected. Whether there are any specific adaptive responses in TRPM8-expressing neurons themselves has been investigated by a number of groups, with varying results. There is little evidence that inflammation alters TRPM8 expression but there is clearly amplified responsiveness to acetone-induced cooling in the intraplantar Complete Freund’s Adjuvant (CFA) model of inflammatory pain [43]. This could potentially reflect increased expression of TRPA1 [73,74] or simply the expected central hypersensitivity to inputs from the TRPM8-mediated innocuous cool afferents [60,61]. In some neuropathic pain models, TRPM8 expression is reported to increase [36,75–77], although little change, or reduced expression, has been reported in others [73,78,79]. Cool allodynia is apparent after nerve injury, and both pharmacological and genetic interventions indicate that this is likely to involve TRPM8 [43,60,61,76–78]. This may, however, reflect simply the TRPM8-mediated reportage of innocuous cooling that, like any other somatosensory input, becomes amplified due to injury-induced central sensitization. Indeed, a recent electrophysiological study reported that acetone-evoked evaporative cooling responses, but not other sensory responses of spinal cord neurons, were inhibited by a selective TRPM8 antagonist in nerve injured but not naive rats [80]. Interestingly, however, in patients with established cool allodynia due to nerve injury, the topical administration of menthol does not aggravate hypersensitivity [81].

Although cooling and mint extracts containing menthol have been widely used for many years due to their soothing, antinociceptive effects, the molecular basis was long unknown [82–85]. The cloning of TRPM8 and its identification in a subset of DRG/TG sensory neurons provided a likely framework. In 2006, TRPM8 was specifically demonstrated for the first time to mediate analgesia due to cooling or the chemical agonists menthol and icilin, applied topically or intrathecally in animal models of both chronic neuropathic pain (CCI, chronic constriction injury) and inflammatory pain [36]. The pharmacological identification of TRPM8 mediation was corroborated by antisense knockdown experiments indicating that active functional TRPM8 was required as opposed to any potential for an effect due to agonist-induced channel desensitization. Both thermal (heat) hyperalgesia and mechanical allodynia were reversed, but cool allodynia was not addressed because of the likelihood of a complex, mixed influence. Interestingly, there was no effect on unsensitized responses in naive animals or in unaffected limbs until much higher concentrations, which produced pronociceptive effects. Experiments investigating the effects of relatively high concentrations of topically applied menthol in naive animals report attenuation of noxious thermal responses, mixed effects on cool/cold responses, and sensitization of innocuous mechanical responses [86], although mediation by TRPM8 was not ascertained, and off-target effects may contribute. The original observations of TRPM8 analgesia were subsequently confirmed in the CCI model of neuropathic pain, in which intrathecal menthol was similarly found to strongly reduce thermal hyperalgesia and mechanical allodynia but increase withdrawal responses from a 4 °C cold plate [77]. Mediator specificity was established by antisense knockdown in this study, too. Interestingly the analgesic effects of TRPM8 agonists were not observed in an alternative neuropathic pain model (SNL, spinal nerve ligation; [87]) in which TRPA1 has been implicated [88]. Evidence for TRPM8-mediated analgesia was also provided in the formalin-induced flinching model, in which cool-induced analgesia was attenuated in TRPM8−/− mice compared to controls [44]. Recent work provides robust support for the idea of TRPM8-mediated analgesia, showing that systemic or topical administration of menthol diminishes pain behavior due to noxious heat, TRPV1 or TRPA1 activators or intraperitoneal acidification as well as attenuating inflammation-induced mechanical hypersensitivity [89]. The critical role of TRPM8 was clearly demonstrated through abrogation of effects in the presence of a highly selective TRPM8 antagonist or in TRPM8−/− mice. Powerful analgesic effects of systemic menthol against formalin-induced flinching and inflammatory hypersensitivity have also been recently described at rather higher dosage levels, although in this case TRPM8-independent mechanisms may also contribute significantly [90]. Key supportive data have also been provided through experiments on targeted ablation of TRPM8-expressing neurons where the cooling-evoked attenuation of mechanical allodynia seen in the CCI neuropathic pain model in control animals was abrogated by toxin-evoked TRPM8 ablation [60]. Corresponding results were seen in TRPM8−/− mice. Further evidence shows that pain state-induced synaptic hypersensitivity not only at the spinal cord but also forebrain levels can be reversed by topical administration of TRPM8 agonist; with the involvement of TRPM8 established through blockade by a highly selective antagonist [91]. Taken together, these observations firmly establish that TRPM8 activation is able to gate-out hypersensitive nociceptive inputs and activation of the CNS in chronic pain states, most likely through the spinal influence of the TRPM8-expressing subset of sensory afferents.

So a case can be made for the use of either antagonists or agonists at TRPM8 in the treatment of pain. Antagonists may be useful to treat the cool allodynia associated with chronic pain states. Effects are likely to be limited to this modality, however, as they would influence only the sensory detection of cool that involves TRPM8 and not the central sensitization that leads to parallel problems of mechanical allodynia and thermal hyperalgesia. Antagonists could potentially be considered for treating acute cold pain in naive subjects, but any evidence to validate this is much less strong than that illustrating the role of TRPM8 in innocuous cool thermosensation. It may well be that other factors play a key part in noxious cold sensing, so any effect of TRPM8 blockade may be less robust; this will become clear in future work. Agonists show great promise in that they are now well documented to produce efficacious analgesia in hypersensitive pain states where they appear to inhibit central sensitization and therefore reverse chronic pain of a number of different modalities. Both neuropathic and inflammatory pain hypersensitivity can be effectively targeted. One caveat with this approach would be that cool allodynia may be exacerbated, although it would be predicted that the analgesic effects of TRPM8 agonists suppressing central hypersensitivity act in opposition to any enhancement of peripheral cool sensing and thereby ameliorate any cool allodynia. Clinical evidence in chronic pain patients supports this idea, as cool allodynia does not seem to be a problematic issue [81,92,93]. Care is also needed in evaluation of the therapeutic window because of the possibility of noxious sensations if supratherapeutic concentrations of agonists are reached. Either strategy (as with any analgesic intervention) could potentially encounter on-target side effects in other tissues or off-target effects due to insufficient pharmacological specificity, a medicinal chemistry issue around the particular pharmacophore utilized. As TRPM8 is expressed in relatively few tissues, the on-target side-effect issue may be relatively unproblematic, especially if agents are applied topically to dermatomes around the site of chronic pain to access selectively the relevant TRPM8 afferents and limit the systemic drug load. For both antagonists and agonists, a further possible issue that would need to be evaluated might be disruption of central thermoregulation, as identified in the case of TRPV1 antagonists.

When antagonists of the noxious-heat sensing channel TRPV1 were tested in vivo as potential analgesics, significant effects on regulation of core body temperature became apparent. TRPV1 antagonists caused hyperthermia, thermogenesis, and vasoconstriction in wild-type but not TRPV1−/− mice [94,95]. Although in contrast to TRPV1, TRPM8 is absent from central thermoregulatory centers in the hypothalamus, potential effects of TRPM8 agonists or antagonists on core temperature have been investigated. Intraperitoneal injection of icilin at high concentrations produces a characteristic acute shivering behavior known as “wet dog shakes,” presumably by stimulating visceral afferents, and this response is attenuated in TRPM8−/− mice [45,96,97]. Systemic or topical administration of menthol or icilin (at relatively high doses) leads to a transient increase in core temperature, presumably an attempt at compensatory thermoregulation [98–103]. Accordingly, systemic TRPM8 antagonists produce a transient reduction in core temperature [61,80,103,104]. In both cases, some studies confirmed lack of effects in TRPM8−/− mice. Whether any significant thermoregulatory changes are observed at the doses therapeutically relevant for the treatment of chronic pain remains to be established.

For a number of years no highly selective antagonists of TRPM8 were available to help validate the inferred role of the channel in thermosensation and modulation of pain processing. Early studies identified that some TRPV1 antagonists also had affinity for TRPM8, providing the first useful but fairly nonselective tools. Capsazepine, BCTC, and SB-452533 were shown to be effective TRPM8 antagonists but with clearly lower potency than at TRPV1, posing difficulties for data interpretation in complex in vivo situations [105,106]. The antifungal agent clotrimazole was identified as a relatively potent TRPM8 antagonist [107], but it additionally blocks K+ channels and activates both TRPV1 and TRPA1. Tryptamine derivatives such as 5-benzoyloxytryptamine were also unexpectedly shown to antagonize TRPM8 but may well impact on 5-HT receptor function, and any potential effects on other pain-relevant channels are unknown [108]. The first clearly selective TRPM8 agonist widely disclosed was AMTB, developed by Bayer [109], although its affinity was still only moderate. Other pharma (including Glenmark, Amgen, Janssen, and Johnson and Johnson) have now developed a number of highly potent and selective TRPM8 antagonists with diverse chemical structures. These include benzothiophene sulphonamides and phosphonates, fused oxazoles and thiazoles, benzimidazoles, fused piperidines, aryl glycines, menthylamines, and ylidenephthalides (although the last two may have some TRPA1/TRPV1 activity) [110–118]. Additional potent and effective TRPM8 antagonists from further structural series have been produced by Pfizer and Takeda [61,80], and even endogenous and plant cannabinoids inhibit at submicromolar concentrations [119], yielding a truly diverse array of pharmacophores as TRPM8 blockers (Figure 14.1).

Figure 14.1 Examples of recently produced TRPM8 antagonists reported to be active at submicromolar concentrations in vitro. Compounds represent several distinct structural series. Although certainly potent TRPM8 antagonists, the pharmacological selectivity profiles are generally not described in detail.